The notion of “friendly” bacteria contributing to good health and well-being was first proposed almost a century ago by Prof. E. Metchnikoff, but it is only in the last two decades that the potential health promoting role of some bacteria has been fully appreciated. Probiotic therapy uses bacterial interference and immunomodulation in the control of several infectious, inflammatory, and immunologic conditions. For instance, there is growing evidence to suggest that while an impoverished or absent gastrointestinal (GI) tract microflora can lead to digestive problems like hypoallergenic intolerance; recolonisation by “friendly” bacteria has the capacity to restore oral tolerance and regain the development of a balanced immune system (Alvarez-Olmos and Oberhelman, 2001; Cross, 2002). While the intricacies of signalling between the de novo colonisers and the immune system are not fully elucidated, it is believed that modulation of the immune response probably occurs through one or a combination of the following mechanisms (Cross, 2002):    1. Localised lactic acid production by probiotics, which may limit the growth of pathogens.    2. Production of anti-pathogenic substances by the probiotic strain e.g. bacteriocins, which are potent bactericidal compounds.    3. Limitation of colonisation by competing for colonisation sites-“competitive exclusion”.    4. Production of immunomodulatory signals by the probiotic strain that stimulate the host immunity sufficiently to afford a degree of enhanced protection.Lactic acid bacteria (LAB), including members of the genera Lactococcus, Lactobacillus, Leuconostoc, Pedicococcus and Streptococcus have been used for millennia in the production of fermented foods. As a result of their history as harmless bacteria, these microorganisms are considered as GRAS (Generally Regarded As Safe) for many applications, including human and animal consumption. In recent years, there has been extensive research into the use of LAB in the control of pathogenic microorganisms, and as health-promoting agents or “probiotics”.
To date, probiotic therapy has mainly been exploited in the treatment of gastrointestinal problems. While initially based on hearsay and tradition, the peer-approved scientific evidence now supporting the protective role of probiotics and in particular the LAB lactobacilli, in the GI tract is immense. Multiple antimicrobial properties of probiotics have been suggested as potential protective factors in the human digestive system against microorganisms such as Escherichia coli, Helicobacter pylori, Salmonella and Listeria species (Alvarez-Olmos and Oberhelman, 2001). For instance, mice which were fed Lactobacillus acidophilus, Lb. casei or a combination of both, prior to oral challenge with Salmonella typhimurium, had reduced pathogen translocation to the spleen and liver, compared with control mice. This resulted in increased survival of mice in the probiotic-fed groups, particularly in the group fed both strains. This study also demonstrated that in the probiotic-fed groups, macrophages had increased phagocytic activity (Perdigon et al., 1990a).
The protective effect of Lb. casei against S. typhimurium, E. coli and Shigella sonnei has also been investigated in mice. Increased protection from oral challenge with the aforementioned pathogens was observed when mice were pre-fed Lb. casei. Additionally, increased IgA levels were observed, and probiotic-fed mice challenged with Shigella had increased anti-Shigella antibody titres in the serum and GI tract compared to the control group (Perdigon et al., 1990b; 1991).
A growing body of evidence, therefore, links increased anti-microbial protection with the enhancement of appropriate immune responses by probiotics. Recently, research has investigated the use of immunomodulatory probiotics as protective agents in the GI tract, and also at other mucosal surfaces. In one such study, mice pre-fed Lb. casei were subjected to an aerosolised challenge of Pseudomonas aeruginosa (Alvarez et al., 2001). The results demonstrated that probiotic feeding increased the clearance rate of P. aeruginosa from the lungs, up-regulated the phagocytic capacity of the alveolar macrophages and increased the levels of IgA in the serum and broncho-alveolar lavage fluid. It is apparent from these results that probiotic feeding can influence immuno-responses in the respiratory tract tissues and that this effect is sufficient to afford protection against bacterial respiratory tract pathogens. Furthermore, Lb. rhamnosus GR-1 and Lb. fermentum RC-14 are well recognised as therapeutic agents in the prevention and treatment of urogenital infections in women. Restoration of a healthy and normal vaginal flora occurs following local application of lactobacilli, demonstrating that probiotics delivered locally, as well as those delivered by the oral route, can provide enhanced protection against pathogens (Reid et al., 2001; Gardiner et al., 2002). Thus, the areas of potential use of probiotics has expanded rapidly in recent decades, and now includes prevention and treatment of diarrhoeal diseases in adults and children, prevention of vaginitis and urinary tract infection in adults, food allergy prevention, and antitumor action in the gut, bladder and cervix (Cross, 2002).
Apart from the obvious benefits of using GRAS organisms for the latter purposes, using Gram-positive bacteria like lactococci, lactobacilli and streptococci has the added advantage that the cell wall of Gram-positive bacteria has been shown to act as an immune-response activator. Another major attraction of using lactic acid bacteria as therapeutic agents stems from their ability to produce bacteriocins, potent anti-microbial peptides (Ross et al., 1999). These peptides kill other microorganisms rapidly by destroying or permeating the microbial membrane and impairing the ability to carry out metabolic processes. Because of their mode of action, these peptides are unlikely to face the same antimicrobial resistance mechanisms that limit current antibiotic use.
Nisin was the first identified bacteriocin derived from fermentation of a lactic acid bacterium, Lactococcus lactis. It is approved for use as a food preservative in the United States, and was awarded GRAS status in the U.S. Federal Register in 1988. It is also approved as a natural food preservative by more than 40 other countries as well as the World Health Organisation and the European Union. In addition to its use as a food additive to inhibit spoilage organisms and pathogens, several studies have investigated its use as a therapeutic agent, in the treatment of such diverse diseases as acne, human gastrointestinal infections and bovine mastitis (Blackburn et al., 1994; Sears et al., 1995). It is currently used as a component of a commercial teat-dip product (CONSEPT®, Babson Bros.).
Lacticin 3147 is a broad-host range bacteriocin also produced from a lactococcal strain, L. lactis DPC3147. It was first identified in an isolate obtained from an Irish kefir-like grain that had been used domestically for the production of buttermilk. It kills all Gram-positive bacteria tested to date, including high profile antibiotic resistant pathogens such as methicillin resistant staphylococci, vancomycin resistant enterococci, and penicillin resistant pneumococci (Galvin et al., 1999) in addition to food poisoning organisms such as Listeria monocytogenes and Clostridium botulinum (Ross et al., 1999). Similar to nisin, it is a member of the family of bacteriocins termed lantibiotics. It is a two-component bacteriocin, with both components required for full activity. Its mode of action involves the formation of pores which, by damaging the membrane of sensitive cells, leak potassium and phosphate ions. Importantly, lacticin 3147 has advantages over nisin as a choice of therapeutic agent, including its effectiveness over a broad pH range (nisin is most effective at acid pH), which suggests additional possibilities in non-acid foods and in biomedical applications (Ross et al., 1999). Lacticin 3147 has already been exploited for a wide range of applications, including use as a powdered biopreservative (Morgan et al., 1999) and in the treatment of bovine mastitis (Ryan et al., 1999; Twomey et al., 2000).
Nutritional competition is established as an important mechanism by which probiotics exert their effect. Suppressive factors such as bacteriocins and toxicity of end metabolic products have also been implicated (Alvarez-Olmos and Oberhelman, 2001; Cross, 2002).
Mastitis is defined as inflammation of the udder and is indicated by increases in Somatic Cell Count (SCC). The SCC is an indication of the levels of neutrophils in the milk, which in turn is an indication of the presence of infection. A normal udder quarter is free from pathogenic bacteria, has very few neutrophils in the milk, and thus, a low SCC (<0.2×106/ml SCC). A rise in SCC usually indicates the presence of an infection.
When a cow has clinical mastitis, the affected quarter may have obvious signs of inflammation-heat, pain and swelling, and the cow may have an elevated body temperature. The SCC is raised above 0.2×106/ml and pathogens may (specific clinical) or may not (non-specific clinical) be detectable. Quarters are also considered clinical, if the milk is visibly abnormal—e.g. clots present, even though there may be no clinical signs on palpation. Clinical mastitis can be classified on the basis of the appearance of the milk from affected quarters. A clinical or subclinical infection is referred to as “Chronic” if it has persisted over a long period and does not respond to antibiotic treatment. Clinical chronic cases are easily identified by the milker. In subclinical cases, the affected udder appears normal but the milk has an elevated SCC (>0.2×106/ml) and pathogens are usually present in the milk. Subclinical chronics are only identified by repeated sampling and laboratory analysis. An EC Council Directive sets out regulations for the hygienic production of milk and dairy products.
Acute and chronic cases are treated routinely with antibiotics. There are cases, however, that do not respond to antibiotic treatment, or cases which respond briefly, and then re-occur, even following repeat administration of antibiotic. Repeated antibiotic administration results in milk loss, as milk must be withheld from the creamery until the milk is free of antibiotic residues.
We have investigated the use of a live culture of L. lactis 3147 in the treatment of bovine mastitis. Use of the bacteriocin-producing culture in place of a concentrated lacticin preparation has certain advantages. Firstly, the producing organism, L. lactis is GRAS, and was isolated from a food source. The use of the live culture for the treatment of mastitis, can be viewed as a prolonged assault on the pathogen—not only is bacteriocin produced in a natural and stable manner, but the culture should also compete with pathogens for colonisation of the teat. Additionally, other antimicrobial substances, such as organic acids, free fatty acids, ammonia, and hydrogen peroxide may also be produced as end products of metabolism. Lastly, the infusion of L. lactis 3147 into the teat duct resulted in an immunomodulatory effect, characterised by a short-lived rise in SCC, with a concomitant reduction or elimination of pathogens; followed by a dramatic improvement in both the clinical outcome and the appearance and the quality of the milk.